Scientists have long sought to improve culturing systems to support medical research in general, and treatment, including cancer treatment, in particular. However, cancer patients are highly individualized in their response to chemotherapies or other anti-cancer regimens. The success of individualized therapy requires accurate pre-testing to determine drug-sensitivity, no matter if the target is DNA, protein, enzyme, hormone or cytoskeleton. In turn, accurate pre-testing requires a tumor sample growing in a microenvironment that is maximally similar to its original condition. Tumor cells grow as ‘seeds’ in their microenvironment, which functions as the ‘soil’. If the ‘soil’ is not well-suited to the growth of the tumor cells, death or biological alterations occur. Although many efforts have been made in this field, a challenge remains regarding the creation of in vitro environments that are suitable for all types of tumors, from different individuals (See Edmondson R et al., Assay & Drug Development Technologies. 2014; 12(4):207-218; Garcia-Posadas et al., Invest Ophthalmol Vis Sci. 2013; 54(10):7143-7152; Ravi M et al., J Cell Physiol. 2015; 230(1):16-26; Thoma C R et al., J Biomol Screen. 2013; 18(10):1330-1337; Tsai M J et al., Hindawi Publishing Corporation, ISRN Biochemistry. 2014; 2014: 1-8; Wu M et al., J Biomech Eng. 2014; 136(2):021011).
For decades, scientists have been using two-dimensional (2D) cell culture studies in attempting to understand tumor biology, function, and pathology. Many chemo-sensitivity tests have been developed based on the 2D culture system. Although these culture systems have produced many important advances, cells grown in 2D conditions can differ considerably in their morphology, cell-cell and cell-matrix interactions and the process of differentiation, from those grown in physiological environments. Three-dimensional (3D) culture models have been developed for cell lines and cells dissociated from tissues (See Birgersdotter A et al., Semin Cancer Biol. 2005; 15(5):405-412; Cukierman E et al., Curr Opin Cell Biol. 2002; 14(5):633-639; Griffith L G et al., Nat Rev Mol Cell Biol. 2006; 7(3):211-224; Nelson C M et al., Annu Rev Cell Dev Biol. 2006; 22:287-309).
Cancer studies have long focused on cloned cancer cells (i.e., established cell lines). Recently, however, the tumor microenvironment has been increasingly recognized as a key contributor to cancer progression and drug resistance (See Heinrich E L et al., Cancer Microenviron. 2012; 5(1):5-18). In the tumor microenvironment (the cellular environment in which the tumor exists) there are blood vessels, immune cells, fibroblasts, signaling molecules, and other mesenchymal cells, as well as the extracellular matrix (ECM). The tumor and its surrounding cells and matrix are closely related and communicate constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth, behavior, and evolution of cancerous cells (See Tsai M J et al., Hindawi Publishing Corporation, ISRN Biochemistry. 2014; 2014:1-8; Wu M et al., J Biomech Eng. 2014; 136(2):021011; Allinen M et al., Cancer Cell. 2004; 6(1):17-32; Bhat R et al., Wiley Interdiscip Rev Dev Biol. 2014; 3(2): 147-163; Bissell M J et al., Cold Spring Harb Symp Quant Biol. 2005; 70:343-356). The tumor microenvironment has also been shown to contribute to tumor heterogeneity (See Wu M et al., J Biomech Eng. 2014; 136(2):021011; Yamada K M et al., Cell. 2007; 130(4):601-610).
In one attempt to mimic the tumor microenvironment, immortalized stroma cells (fibroblast, endothelial) were mixed with cancer cells in the culture (See Thoma C R et al., J Biomol Screen. 2013; 18(10): 1330-1337; Fu W et al., Chung-Kuo Hsiu Fu Chung Chien Wai Ko Tsa Chih/Chinese Journal of Reparative & Reconstructive Surgery. 2014; 28(2):179-185). A more recent study has shown that using irradiated stroma cells as the feeder layer in cultures can help tumor cells re-build their histologic structure (See Saenz F R et al., PLoS One. 2014; 9(5):e97666). But because the source of these stroma cells is either biologically different from the tumor cells or has lost the function to communicate with tumor cells (after irradiation), these artificial microenvironments do not closely represent conditions in the patient.
In addition to cellular effects, molecular biochemistry plays another important role in the microenvironment of a tumor. The pleiotropic nature of cytokines in the microenvironment contributes to promoting cancer cell proliferation, bypassing apoptosis, inducing the EMT (epithelial-mesenchymal-transition) of cancer cells, enhancing chemokines to recruit immune suppressor cells that aggregate around the tumor, and even driving the development of drug resistance (See Shain K H et al., Expert Rev Hematol. 2009; 2(6):649-662). Increasing evidence demonstrates that a variety of inflammatory mediators from cancer and tumor-infiltrating cells, such as IL-1, IL-6, and IL-8, facilitate the development of a tumor microenvironment that favors tumor cell proliferation, motility and invasion, and thereby increases their metastatic potential (See O'Callaghan D S et al., J Thorac Oncol. 2010; 5(12):2024-2036; Gilbert C A et al., Annu Rev Med. 2013; 64:45-57). All of these in vivo factors make the selection of a cancer therapy for an individual much more complicated and challenging.
Human blood supplies in vivo bring large amount of nutrients as well as necessary cytokines, chemokines, growth factors, and hormones supporting tumor growth. In current 2D and 3D culture systems, these nutrients are supplied by 5-20% fetal bovine serum (FBS). Although cytokines/growth factors have been manually added into certain culture systems to support and/or maintain the growth of tumor cells in vitro (See Saenz F R et al., PLoS One. 2014; 9(5):e97666; Kobayashi H et al., Int J Oncol. 1997; 11(3):449-455; Kobayashi H, Recent Results Cancer Res. 2003; 161:48-61; Kobayashi H, Methods Mol Med. 2005; 110:59-67; Nakagawa T et al., Gan To Kagaku Ryoho. 2004; 31(13):2145-2149), these artificial conditions could be very different from those in an individual patient. The use of human blood to culture human cells has also been researched; however, previous studies have employed pooled normal human serum (commercially provided) rather than individualized (See Isaac C et al., Rev Bras Cir Plast. 2011; 26 (3):379-384). Significantly different gene/protein expressions are observed between cells cultured with FBS versus human serum. Even within an individual, blood chemistry varies with a patient's physiological condition, as well as the type and stage of their disease (See Whitney A R et al., Proc Natl Acad Sci USA. 2003; 100(4): 1896-1901; Baine M J et al., Methods Mol Biol. 2013; 980: 157-173; Chen Y et al., J Cancer Res Clin Oncol. 2014).
Because in vitro cell culture systems lack the relevant physiological characteristics necessary for drug evaluation, the investigation of efficacy through in vivo models (e.g., murine models) has been widely accepted. However, implanting a human tumor into an animal results in an abrupt change in the microenvironment of the tumor, forcing the tumor cells to attempt to communicate with stroma cells from a different species. In addition, the high financial cost of animal experimentation mitigates against its widespread clinical use.
Tumors shed cells that can enter the bloodstream. These are called circulating tumor cells (CTCs) which can take root elsewhere, causing the spread of the cancer (See Castle J et al., The Breast. 2014; 23(5):552-560; Kolostova K et al., Am J Transl Res. 2015; 7(7):1203-1213; Eliasova P et al., Folia Histochem Cytobiol. 2013; 51(4):265-277; Friedlander T W et al., J Clin Oncol. 2014; 32(11):1104-1106; Friedlander T W et al., Pharmacol Ther. 2014; 142(3):271-280). There is considerable interest in CTC research and technologies for their potential use as cancer biomarkers that may enhance cancer diagnosis and prognosis, facilitate drug development, and improve the treatment of cancer patients. (See Harouaka R et al., Pharmacol Ther. 2014; 141(2):209-221). The isolation and analysis of CTCs are useful methods for tracking how cancers evolve during disease progress and therapy. However, because these cells occur in very low numbers and circulate through the body, isolating CTCs from the blood of cancer patients has been a technical challenge (See Castle J et al., The Breast. 2014; 23(5):552-560; Kolostova K et al., Am J Transl Res. 2015; 7(7):1203-1213; Eliasova P et al., Folia Histochem Cytobiol. 2013; 51(4):265-277; Friedlander T W et al., J Clin Oncol. 2014; 32(11): 1104-1106; Friedlander T W et al., Pharmacol Ther. 2014; 142(3):271-280).
It is well-known that, to date, drugs are usually much more effective in experimental studies (both in vitro and in vivo) than in actual clinical practice. An approach for individualized drug-sensitivity testing was created by Dr. Hisayuki Kobayashi, a technique which has undergone clinical trials (See Kobayashi H, Recent Results Cancer Res. 2003; 161:48-61; Kobayashi H, Methods Mol Med. 2005; 110:59-67; Higashiyama M et al., Lung Cancer. 2010; 68(3):472-477; Higashiyama M et al., J Thorac Dis. 2012; 4(1):40-47; Higashiyama M et al., Ann Thorac Cardiovasc Surg. 2008; 14(6):355-362; Kawamura M et al., Cancer Chemother Pharmacol. 2007; 59(4):507-513; Nagai N et al., Anticancer Drugs. 2005; 16(5):525-531; Naitoh H et al., Gastric Cancer. 2014; 17(4):630-637; Tanioka M et al., Exp Ther Med. 2010; 1(1):65-68). This technique is named CD-DST (the collagen gel droplet embedded culture-drug sensitivity test (See Kobayashi H et al., Int J Oncol. 1997; 11(3):449-455; Kobayashi H et al., Int J Oncol. 1997; 11(3):449-455)). The working principle of this technique is to culture cancer cells from individual patients in a 3D environment for a relatively short time (15-20 days), but long enough for drug testing. Tumors from different patients are cultured under the same artificial conditions, basically with a nutrient supply consisting of 10% FBS-complemented commercial culture media plus some growth factors (See Saenz F R et al., PLoS One. 2014; 9(5):e97666). The drug sensitivity testing is performed in a microenvironment that is far different from the tumor's natural condition. Accordingly, the results of testing with this culture system may not accurately predict the response of the tumor when the drug is administered to the patient. Additionally, use of 2% autologous serum in a culture medium also having 8% FBS has also been described (Majumder B et al., Nature Communications 2015; 6:1-14). Commercial growth factors and special antibodies were also added into these cultures. Such a culture medium is far different from a patient's real condition.
Thus, there is a need for improved culture methods that more closely replicate in vivo conditions.
This background information is provided for informational purposes only. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the present invention.